The expression level of HLA class-I proteins is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences among people with regard to the steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult because of the tremendous polymorphism of HLA. Most individuals express six classical HLA alleles (i.e., heterozygous at HLAA, HLAB, and HLAC) in addition to HLAE, so detecting a particular locus with specificity is challenging. Even if antibodies specific to molecules from each locus can be identified, their binding cannot simply be compared because of differences in affinity for their respective antigens. Relative expression levels of the classical HLA class-I loci are of particular interest on HIV-infected cells, because HIV encodes the Nef protein, which downregulates HLAA and HLAB. Nef has multiple functions but, specifically, downregulation of HLA/MHC was shown to be significant in vivo. Because HLAA and HLAB are not reduced with equal efficiency by HIV, and Nef does not modulate HLAC, it is not clear which HLA locus dominates on HIV-infected cells . We used two independent approaches, flow cytometry and mass spectrometry (MS), to determine the relative expression levels of HLA class-I proteins on normal and HIV infected primary cells. PBLs from normal donors showed that HLAA and HLAB proteins are expressed at similar levels, which are 13 to 18 times higher than HLAC by flow cytometry and 4 to 5 times higher than HLAC by mass spectrometry; these differences may reflect variation in the conformation or location of proteins detected. Primary CD4 T cells infected with HIV in vitro were also studied because HIV downregulates selective HLA types. HLAA and HLAB were reduced on HIV infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLAA to be 1 to 3 times higher and HLAB to be 2 to 5 times higher than HLAC by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV infected cells. The large differences in expression levels observed between HLA class I loci are likely to be functionally significant. Higher HLA expression levels are known to more efficiently initiate CTL responses and modulate the cytokines that CTLs secrete. Further, some inhibitory receptors (such as LILRB1/2 and KIR3DL1) recognize antigens from multiple HLA loci, so allotype-specific expression levels may affect the innate-immune response, as well as the acquired-immune response. Given the accumulating data pointing to a significant impact of differential allotype-specific and locus-specific expression levels on the immune response, it is important to define this property for each HLA locus to determine its potential effect across human diseases. The highly polymorphic HLA class I and class II genes map to the human MHC and encode molecules that contribute to both the adaptive and innate immune responses. Genome-wide association studies have identified the MHC as the most rich 4 Mb region of the genome in terms of association with virtually all types of complex human disease, and for many of these diseases, they highlight this region as the most important in determining disease risk genome wide. Higher HLAC expression levels have been associated with enhanced human immunodeficiency virus (HIV) control, greater odds of generating HLAC restricted cytotoxic T lymphocyte (CTL) responses to the virus, and stronger immune pressure on HIV as measured by viral escape mutations. HLA-C expression levels also associate with risk of Crohn's disease, but in this case, low expression appears to confer protection. Opposing effects of expression levels on various diseases may indicate selection pressure to maintain differential expression across HLA allelic lineages, a model supported by genetic evolution analyses of the HLAC locus. With this in mind, we recently examined the expression levels of allelic lineages at the HLAA locus in a sample of 216 European Americans using a real-time polymerase chain reaction assay, and observed a gradient of expression that associates with HLAA allelic lineage. DNA methylation of the HLAA gene appears to contribute to the variation in HLAA mRNA expression levels, as a significant inverse correlation was observed between HLAA mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor. Further, deep-sequencing and immunoprecipitation assays revealed allelic lineage-specific methylation patterns within the HLA-A promoter region where increased DNA methylation levels correlated significantly with reduced HLA-A expression levels. These data demonstrate HLA-A allelic lineage-specific variation in expression levels, which is due at least in part to differential DNA methylation patterns where low HLA-A expressing lineages have higher DNA methylation levels, and vice versa, in healthy European American (EA) donors. This mode of transcriptional regulation is distinct from HLA-B and HLA-C, as all HLA-B and -C lineages are completely unmethylated in spite of having the similar CpG target sites for methylation as HLA-A, further distinguishing the evolutionary history of the classical HLA class I loci. Several mechanisms are likely to contribute to the variation in expression levels of HLA-A lineages, and characterizing these mechanisms may present the potential for HLA-A-specific regulation of expression levels as drug targets.